Ergosteroid and Phenolic Constituents from the Mushroom Sanghuangporus vaninii with Anti-Inflammatory Activity

Abstract

Sanghuangporus vaninii (Ljub.) is an edible and medicinal macrofungus that has become the main strain for artificial cultivation of Sanghuang. In this study, twenty-six compounds (1–26), including five previously undescribed ergosterols, named sanghusterols A–E (1–5), were isolated from the fruiting bodies of S. vaninii. Their structures were elucidated by spectroscopic methods and electronic circular dichroism (ECD) calculations. Compounds 1, 15, 17, 21 and 25 exhibited potent inhibitory activity against NO production with the IC₅₀ value of 8.3–14.8 μM and dose-dependently decreased iNOS and COX-2 protein expression in RAW264.7 cells. Molecular docking studies confirmed the capacity of compounds 1, 15, 17, 21 and 25 to interact with iNOS and COX-2 proteins. These findings may provide a solid phytochemical and pharmacological basis for developing the mushroom as potential anti-inflammatory agents.

1. Introduction

Chronic inflammation is a key driver in the initiation, progression, invasion, and metastasis of tumors. In order to clear tumor cells, the immune system is activated and macrophages are stimulated, leading to the production of pro-inflammatory mediators including cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α [1]. The genus Sanghuangporus comprises edible and medicinal fungi that have been used in China for over 2000 years to treat various diseases [2]. They contain many chemical components with various pharmacological effects, including anti-tumor, antioxidant, hypoglycemic, and anti-inflammatory properties, with their anti-tumor activity being particularly notable, and they are therefore known as ‘forest gold’ [3]. Sanghuangporus vaninii (Ljub.) can be artificially cultivated in the bag and cut log, the fruiting bodies of which are rich in sterols, polysaccharides, flavonoids, polyphenols, etc. [4]. Among sterol compounds, ergosterol constitutes the largest proportion. Through structural modifications such as ring cleavage, rearrangement, and degradation, ergosterol can form novel carbon skeletons and exhibit diverse biological activities, particularly its anti-inflammatory activity, which even surpasses that of certain pharmaceutical drugs [5,6].

To elucidate the anti-tumor active components of S. vaninii while continuing our ongoing research on extracting novel bioactive substances from large fungi [7,8,9,10,11], a chemical investigation on cultivated S. vaninii was carried out, leading to the isolation of eighteen steroids (1–18), including five new ones (1–5), along with other eight known compounds (19–26) (Figure 1). The inhibitory effects of compounds 1–13, 15–21, 25 and 26 on anti-inflammatory activity were evaluated, and compounds 1, 15, 17, 21 and 25 significantly inhibited the release of NO in LPS-stimulated RAW264.7 cells, with IC₅₀ values ranging from 8.3 to 14.8 μM. Moreover, they blocked the expression of pro-inflammatory cytokines (iNOS and COX-2) in a dose-dependent manner. 1D/2D NMR and HRESIMS spectra of compound 1–5 (Figures S1–S35), ¹H and ¹³C NMR spectra of compound 6–21 and 24–26 (Figures S36–S73), Dose–response curve for the compounds 1, 15, 17, 21 and 25 (Figures S74–S78).

2. Results

2.1. Structure Elucidation

Compound 1 was isolated as a white powder, and its molecular formula was determined as C₂₈H₄₂O by HRESIMS and NMR data. The ¹H NMR data displayed six methyl groups [δ_H 0.59 (3H, s), 0.82 (3H, d, J = 7.1 Hz), 0.84 (3H, d, J = 7.1 Hz), 0.92 (3H, d, J = 6.8 Hz), 1.02 (3H, d, J = 6.5 Hz), and 1.25 (3H, s)], one oxygenated methine 3.61 (1H, m), and five olefinic protons [δ_H 5.16 (1H, dd, J = 15.3, 8.2 Hz), 5.23 (1H, dd, J = 15.3, 7.4 Hz), 5.40 (1H, d, J = 5.9 Hz), 5.52 (1H, d, J = 6.4 Hz), and 5.68 (1H, d, J = 5.9 Hz)] (

Table 1. ¹H NMR data for compounds 1–5 (400 MHz, J in Hz).

No.	1 a	2 a	3 b	4 b	5 b
1	1.51 (m), 1.72 (m)	1.51 (m), 1.72 (m)	1.70 (m), 1.88 (m)	1.60 (m),1.50 (m)	1.75 (m), 1.42 (m)
2	1.41 (m), 1.74 (m)	1.88 (m), 1.28 (m)	1.90 (m), 1.55 (m)	1.78 (m), 1.44 (m)	1.71 (m), 1.28 (m)
3	2.17 (m), 2.33 (m)	4.07 (m)	3.92 (dt, 16.1, 5.7)	3.98 (m)	3.96 (dt, 15.7, 5.7)
4	5.52 (d, 6.4)	1.71 (m),1.55 (m)	2.23 (m), 1.43 (m)	2.10 (m), 1.70 (m)	1.94 (m), 1.58 (m)
6	5.68 (d, 5.9)	4.26 (brs)	4.03 (brs)	3.17 (d, 4.9)	3.55 (d, 2.4)
7	5.40 (d, 5.9)	5.15 (brs)	5.12 (brs)	5.42 (d, 4.9)	4.13 (d, 2.4)
9				2.02 (m)	2.32 (m)
11	3.61 (m)	5.68 (m)	5.62 (d, 6.3)	1.56 (m)	1.60 (m)
12	2.47 (m), 2.38 (m)	2.35 (m), 2.18 (m)	2.38 (m), 2.18 (m)	2.10 (m), 1.33 (m)	2.02 (m), 1.20 (m)
14	2.22 (m)	2.23 (m)	2.37 (m)	1.96 (m)
15	1.46 (m), 1.79 (m)	1.80 (m), 1.46 (m)	1.87 (m), 1.48 (m)	1.62 (m), 1.52 (m)	2.41 (m)
16	1.70 (m), 1.92 (m)	2.00 (m), 1.36 (m)	2.03 (m), 1.38 (m)	1.95 (m), 1.34 (m)	1.75 (m), 1.46 (m)
17	1.30 (m)	1.34 (m)	1.38 (m)	1.33 (m)	1.20 (m)
18	0.59 (s)	0.57 (s)	0.60 (s)	0.63 (s)	0.95 (s)
19	1.25 (s)	0.97 (s)	1.10 (s)	1.00 (s)	1.00 (s)
20	2.04 (m)	1.38 (m)	1.42 (m)	1.40 (m)	2.16 (m)
21	1.02 (d, 6.5)	0.97 (d, 6.6)	0.98 (d, 6.6)	0.98 (d, 6.6)	1.09 (d, 6.6)
22	5.16 (dd, 15.3, 8.2)	1.47 (m), 0.99 (m)	1.50 (m), 1.02 (m)	1.46 (m), 1.00 (m)	5.22 (dd, 15.4, 7.8)
23	5.23 (dd, 15.3, 7.4)	1.43 (m), 0.98 (m)	1.48 (m), 1.02 (m)	1.43 (m), 0.99 (m)	5.24 (dd, 15.4, 7.8)
24	1.87 (m)	1.21 (m)	1.25 (m)	1.24 (m)	1.86 (m)
25	1.47 (m)	1.58 (m)	1.60 (m)	1.60 (m)	1.47 (m)
26	0.82 (d, 7.1)	0.89 (d, 7.0)	0.91 (d, 7.0)	0.90 (d, 7.0)	0.86 (d, 6.8)
27	0.84 (d, 7.1)	0.82 (d, 7.0)	0.84 (d, 7.0)	0.83 (d, 7.0)	0.86 (d, 6.8)
28	0.92 (d, 6.8)	0.82 (d, 6.8)	0.84 (d, 6.8)	0.83 (d, 6.8)	0.94 (d, 6.9)
6-OCH3				3.39 (s)
7-OCH3					3.22 (s)

^a Recorded in CDCl₃. ^b Recorded in methanol-d₄.

). The ¹³C NMR spectrum revealed 28 carbon resonances comprising six methyl groups, six methylenes, eleven methines (including an oxygenated carbon), and five quaternary carbons (

Table 2. ¹³C NMR data for compounds 1–5 (100 MHz).

No.	1 a	2 a	3 b	4 b	5 b
1	38.5, CH2	30.3, CH2	31.2, CH2	33.6, CH2	30.2, CH2
2	23.0, CH2	32.0, CH2	31.7, CH2	31.7, CH2	31.6, CH2
3	43.0, CH2	68.4, CH	67.8, CH	68.3, CH	66.3, CH
4	122.6, CH	29.5, CH2	39.1, CH2	40.7, CH2	39.7, CH2
5	141.5, C	78.8, C	77.0, C	78.0, C	76.7, C
6	118.4, CH	72.5, CH	71.6, CH	83.9, CH	75.2, CH
7	115.8, CH	122.8, CH	121.7, CH	116.0, CH	80.4, CH
8	135.6, C	140.4, C	142.1, C	144.5, C	122.0, C
9	144.4, C	138.2, C	138.7, C	44.8, CH	36.6, CH
10	39.5, C	44.2, C	41.6, C	38.2, C	40.5, C
11	72.4, CH	123.5, CH	123.5, CH	23.0, CH2	19.0, CH2
12	41.7, CH2	43.8, CH2	43.7, CH2	40.8, CH2	37.3, CH2
13	42.3, C	43.6, C	43.6, C	44.7, C	43.5, C
14	51.2, CH	52.3, CH	52.2, CH	53.0, CH	153.3, C
15	29.1, CH2	24.2, CH2	24.2, CH2	24.0, CH2	25.2, CH2
16	32.4, CH2	29.4, CH2	29.5, CH2	28.9, CH2	27.1, CH2
17	56.3, CH	57.4, CH	57.4, CH	57.3, CH	57.2, CH
18	11.7, CH3	11.7, CH3	11.8, CH3	12.4, CH3	16.7, CH3
19	30.6, CH3	26.1, CH3	25.1, CH3	18.8, CH3	16.9, CH3
20	40.6, CH	37.6, CH	37.6, CH	37.9, CH	39.2, CH
21	20.8, CH3	19.1, CH3	19.1, CH3	19.5, CH3	20.5, CH3
22	135.5, CH	34.8, CH2	34.7, CH2	34.8, CH2	135.4, CH
23	132.2, CH	31.7, CH2	31.7, CH2	31.7, CH2	132.1, CH
24	42.9, CH	40.4, CH	40.4, CH	40.4, CH	43.0, CH
25	33.2, CH	32.7, CH	32.7, CH	32.7, CH	33.0, CH
26	20.1, CH3	20.9, CH3	20.9, CH3	20.9, CH3	19.1, CH3
27	19.8, CH3	18.0, CH3	18.0, CH3	18.0, CH3	18.7, CH3
28	17.7, CH3	15.9, CH3	15.9, CH3	15.9, CH3	16.8, CH3
6-OCH3				58.2, CH3
7-OCH3					53.7, CH3

^a Recorded in CDCl₃. ^b Recorded in methanol-d₄.

). A further comprehensive analysis of the 2D NMR spectra is required. The ¹H-¹H COSY correlations (Figure 2) revealed the presence of four spin-coupling systems in 1, including H₂-1/H₂-2/H₂-3/H-4; H-6/H-7; H-11/H₂-12; and H-14/H₂-15/H₂-16/H-17/H-20/(H₃-21)/H-22/H-23/H-24(H₃-28)/H-25/H₃-26(H₃-27). The HMBC correlations (Figure 2) from H₃-19 to C-1, C-9, and C-10; from H-4/H-6 to C-5; from H-7 to C-8/C-14; from H-11 to C-8/C-9; and from H₃-18 to C-12/C-13/C-17 established the carbon connectivity, and the gross structure of 1 is much similar to the ergosteroid skeleton as ergosta-4,6,8,22E-tetraen-11β-ol from Coprinus setulosus [12]. The only difference observed in the ¹³C NMR spectrum was that the chemical shift of C-19 in 1 (δ_C 30.6) moved downfield compared to C-19 in ergosta-4,6,8,22E-tetraen-11β-ol (δ_C 22.4). The NOE correlations (Figure 3) of H-11/H₃-18, H-12b/H₃-18, H-12a/H₃-19, H-12a/H-17, and H-17/H-14 demonstrated that H₃-19, 11-OH, H-14, and H-17 were α-oriented, while H₃-18 was β-oriented. The double bond Δ^22,23 was determined as E-configuration by the large coupling constant (J = 15.3 Hz). The experimental and calculated ECD spectra demonstrated a high degree of congruence (Figure 4), suggesting that the absolute configuration of 1 is 10S,11S,13R,17R,20R,24R, and named as (22E,24R,10S)-ergosta-4,6,8(9),22-tetraen-11α-ol.

Compound 2 was obtained as a white powder. Its molecular formula was determined to be C₂₈H₄₆O₃ by HRESIMS data, which also indicated six degrees of unsaturation. The 1D and 2D NMR spectra revealed that compound 2 contained 28 carbons, which were classified as six methyl groups, eight methylenes, nine methines and five quaternary carbons (Table 1 and Table 2). This suggests that 2 may be an ergosteroid. The characteristic signals of two singlet methyl groups at δ_H 0.57 (3H, s) and 0.79 (3H, s), and four doublet methyls at δ_H 0.82 (3H, d, J = 7.0 Hz), 0.82 (3H, d, J = 6.8 Hz), 0.89 (3H, d, J = 7.0 Hz), and 0.97 (3H, d, J = 7.0 Hz), as observed in the ¹H NMR, further indicated the ergosteroid skeleton of 2. Combining with 2D NMR data (Figure 2), the structure of compound 2 was similar to the gross structure of (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6β-triol [13]; the only difference was that 22,23-position carbons of compound 2 were an ethylidene group. By combining NOESY spectrum and the biosynthetic pathway [13], the relative configuration of compound 2 was determined to be rel-3S,5S,6S,10R,13R,17R,20R,24S (Figure 3). The absolute configuration of 6 was determined by comparing the experimental and calculated ECD spectra (Figure 4). Therefore, compound 2 was elucidated as (24R)-ergosta-7,9(11)-diene-3β,5β,6α-triol.

Compound 3 had the same molecular formula (C₂₈H₄₆O₃) as 2 according to their (+)-HRESIMS and the ¹³C NMR data. Through scrutiny of the 1D and 2D NMR data of 3 and 2, only slight differences were observed for the vicinal protons and carbons adjacent to the C-5 CH group. The same HMBC correlations were reported for both compounds, implying that they are stereoisomers (Figure 2). Similar NOESY patterns and ECD data for compounds 3 and 2 indicated that the only difference between them is the configuration of C-5 (Figure 3 and Figure 4). Therefore, compound 3 was assigned as a 5-epimer of 2 and named (24R)-ergost-7,9(11)-dien-3β,5α,6α-triol.

The molecular formula of (24R)-6β-methoxyergosta-7-ene-3β,5α-diol (4), white powder, was determined to be C₂₉H₅₀O₃ (five degrees of unsaturation) based on its HRESIMS data at m/z 469.3653 [M + Na]⁺ (calcd for C₂₉H₅₀O₃Na, 469.3652). The ¹H and ¹³C NMR data for 4 were greatly similar to those for 3 with the absence of a double bond at C-9/C-11 positions (δ_C 44.8 and 23.0) and the additional presence of a methoxy group attached at C-6 in 4 (δ_H 3.39; δ_C 58.2) (Table 1 and Table 2). This conclusion was supported by ¹H-¹H COSY correlation between H-9 (δ_H 2.02) and H₂-11 (δ_H 1.56) and HMBC correlations from H-9 (δ_H 2.02) to C-7 (δ_C 116.0), C-5 (δ_C 78.0), and 6-OCH₃ (δ_H 3.39) to C-6 (δ_C 83.9) (Figure 2). The relative configuration of 4 was consistent with 3, as evidenced by NOESY correlations of H-3/H-6, H-6/H-14, H-9/H-14, H-14/H-17, and H₃-18/H₃-19 (Figure 3). The calculated ECD spectrum of 3S,5S,6S,10R,13R,17R,20R,24S matched with the experimental ECD data (Figure 4).

Compound 5 was acquired as a white powder and exhibited a [M + Na]⁺ peak at m/z 483.3447 in the HRESIMS spectrum, disclosing its molecular formula to be C₂₉H₄₈O₄. Comprehensive analysis of the ¹H and ¹³C NMR spectra (Table 1 and Table 2) for 5 disclosed that they were quite similar to those of the reported metabolite (22E,24R)-6β,7α-dimethoxyergosta-8(14),22-diene-3β,5α-diol (6) [13] except for signals of a methoxy group. Only one methoxyl (δ_H 3.22, δ_C 53.7) was observed in compound 5. Through further detailed analysis of 2D NMR data, the only difference between 5 and 6 was that the methoxy group at C-6 disappeared, which was corroborated by the HMBC relationships from H-6 to C-8 and C-10, from 7-OCH₃ to C-7, and from H-7 to C-5 and C-14 (Figure 2). The smaller coupling constants (J_H6,H7 = 2.4 Hz) indicated a cis configuration, while the NOE-related signals between H-6 and H₃-19 suggested both adopt the β configuration (Figure 3). Similarly, the double bond Δ^22,23 was determined to be the E configuration, as indicated by the large coupling constants (J = 15.4 Hz). The absolute configuration in 5 was then affirmed as 3S,5R,6S,7S,10R,13R,17R,20R,24R according to the perfect similarity between the calculated and the experimental ECD spectra (Figure 4). Therefore, the structure of 5 was verified as (22E,24R)-7α-methoxyergosta-8(14),22-diene-3β,5α,6α-triol.

Twenty-one known compounds were determined as (22E,24R)-6β,7α-dimethoxyergosta-8(14),22-diene-3β,5α-diol (6) [13], ergosterol (7) [13], penicisterol F (8) [14], (22E,24R)-ergosta-7,9(11),22-triene-3β,5β,6α-triol (9) [13], 6β-methoxyergosta-7,9(11),22E-triene-3β,5α-diol (10) [15], 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol (11) [16], ergosta-7,22-dien-6β-methoxy-3β,5α-diol (12) [17], 3β,5α,9α-trihydroxyergosta-7,22-dien-6-one (13) [18], (22E,24R)-ergosta-7,22-dien-3β,5α-diol-6-one (14) [13], 3β,5α,9α,14α-tetrahydroxy-(22E,24R)-ergosta-7,22-dien-6-one (15) [19], (22E,24S)-6-O-methyl-24-methylcholesta-7,22-diene-3β,5α,6β,9α-tetrol (16) [20], 3β-hydroxy-5,9-epoxy-(22E,24R)-ergosta-7,22-dien-6-one (17) [13], (22E,24R)-ergosta-7,22-dien-3β,5α-diol-6,5-olide (18) [21], (2R)-1,2-ethanediol, 1-(3-ethylphenyl)-, 1,2-dibenzoate (19) [22], (2R)-1,2-ethanediol, 1-(4-ethylphenyl)-, 1,2-dibenzoate (20) [22], inoscavin B (21) [23], (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one (22) [24], 3,4-dihydroxybenzoate (23) [25], 3-N-acetyl-β-oxotryptamine (24) [26], cinnamic acid bornyl ester (25) [27], and chrysogeside E (26) [28] by comparing their spectroscopic data with those reported in the literature.

2.2. Anti-Inflammatory Assays

Compounds 1–13, 15–22 and 25 were evaluated for their anti-inflammatory activity in a classical model of LPS-induced NO release in RAW264.7 cells. All tested compounds had no obvious effect on cell viability at a concentration of 20 μM. As shown in

Table 3. Inhibitory effects of compounds 1–13, 15–21, 25 and 26 on NO production in LPS-induced RAW 264.7 cells.

Comp.	IC50 (μM) ± SD (μM)	Comp.	IC50 (μM) ± SD (μM)
1	10.1 ± 0.1	13	NA
2	NA	15	8.3 ± 0.5
3	NA	16	NA
4	NA	17	10.6 ± 0.4
5	NA	18	NA
6	NA	19	NA
7	NA	20	NA
8	NA	21	8.9 ± 0.2
9	NA	25	14.8 ± 0.4
10	NA	26	NA
11	NA	DEX a	9.7 ± 0.2
12	NA

^a Positive control. NA: no active.

, compounds 1, 15, 17, 21 and 25 exhibited inhibitory effects against LPS-induced NO production in RAW264.7 macrophages with IC₅₀ values ranging from 8.3 to 14.8 μM, being comparable to the well-known NO inhibitor, dexamethasone (IC₅₀ = 9.7 μM) [29]. Compounds 15, 17, 21 and 25 all contain an α,β-unsaturated ketone structural moiety. As Michael acceptors, these moieties could potentially react with nucleophiles involved in NO biosynthesis in biological systems. As shown in Figure 5, a Western blotting assay revealed that compounds 1, 15, 17, 21 and 25 dose-dependently inhibited LPS-induced iNOS and COX-2 expression in RAW264.7 macrophages at 5, 10, and 20 μM. Molecular docking is a theoretical simulation method that studies intermolecular interactions and predicts their binding patterns and affinity. The binding modes and forces between the compounds 1, 15, 17, 21 and 25 and inflammatory factors (iNOS and COX-2) were investigated using AutoDockTools software. The results of the molecular docking analysis indicated that compounds 1, 15, 17, 21 and 25 exhibited a high affinity for iNOS and COX-2, with a binding energy arrange from −8.8 to −7.2 kcal/mol (Figure 6 and

Table 4. Binding energy of compounds 1, 15, 17, 21, and 25 with iNOS and COX-2.

Compound	Binding Energy
	iNOS	COX-2
1	−7.7	−8.2
15	−8.1	−8.4
17	−7.2	−8.8
21	−7.2	−8.2
25	−7.9	−8.2

).

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotation (OR) was measured on a Rudolph Autopol III automatic polarimeter at 20 °C (Rudolph Research Analytical, Hackettstown, NJ, USA). Ultraviolet (UV) and experimental Circular Dichroism (ECD) spectra were recorded on a Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, UK). IR spectra were obtained on Bruker TENSOR 27 spectrometer (Bruker, Mannheim, Germany). NMR spectra were recorded on a Bruker AM-400 NMR spectrometer (Bruker, Mannheim, Germany). HRESIMS data was completed by a LC-30A + TripleTOF5600+(AB SCIEX, Framingham, MA, USA).

3.2. Fungal Material

The bag-cultivated sporocarps of S. vaninii were collected from Yulin City, Shaanxi Province, in December 2022. The strain was identified by Pro. Shuang Tian Du of College of Life Sciences and preserved in the key laboratory of chemical biology of natural products, Northwest A&F University, Yangling, Shaanxi.

3.3. Extraction and Isolation

The 20 kg dried fruiting bodies of S. vaninii were pulverized, and the powder was extracted with methanol by heating with reflux. The methanol extract was evaporated to afford the crude product (1.17 kg). The crude extract was dissolved in distilled water (2.0 L) and then extracted three times with petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EA), and n-butanol (n-BuOH), successively. The EA and n-BuOH extracts (143.0 g) were subjected to a small pore resin coagulant gel column (MCI) with MeOH/H₂O (40%, 60%, 80%, 100%) to get four fractions (Fr.A~Fr.D). Fr.A was separated by silica gel CC eluting stepwise with DCM-MeOH (40:1 → 2:1) to give Fr.A1-A3, and then Fr.A1 and A2 were further purified by Sephadex LH-20 (DCM/CH₃OH, v/v, 1:1) and semi-prep RP-HPLC (CH₃OH/H₂O, 45%) to yield compounds 21 (23.8 min, 2.6 mg), 23 (28.6 min, 3.0 mg), 24 (25.5 min, 2.0 mg), and 25 (29.0 min, 2.5 mg). Fr.D was applied to a silica gel CC (300–400 mesh) eluting with CH₂Cl₂–MeOH (100:1–5:1, v/v) to afford six subfractions (Fr. D1~D6). Fr. D1 was separated with silica gel CC (PE/EA, 20:1 to 2:1) to obtain three fractions (Fr.D1-1~Fr.D1-3), and Fr.D1-3 was further purified by semipreparative HPLC (MeOH/H₂O, 76:24, v/v) to yield compounds 1 (33.1 min, 3.8 mg) and 6 (36.1 min, 29.0 mg). Fr.D1-1 was chromatographed with silica gel CC (PE–EA, 20:1 to 2:1) to give three fractions (Fr.D1-1-1~Fr.D1-1-3), then Fr.D1-1-2 and Fr.D1-1-3 were purified by RP-HPLC (MeOH/H₂O, 76:24, v/v) to afford compounds 22 (24.2 min, 1.5 mg), 19 (28.3 min, 4.7 mg) and 20 (33.1 min, 2.9 mg). Fraction D3 was subjected to silica gel CC (300–400 mush) eluting with DCM/MeOH (from 20:1 to 2:1, v/v) to obtain four fractions (Fr.D3-1~Fr.D3-4). After that, fraction D3-1 was separated by prep-HPLC (MeOH/H₂O, 76:24, v/v), which produced compounds 3 (16.1 min, 4.8 mg), 8 (19.9 min, 21.0 mg), 9 (29.4 min, 3.3 mg), 10 (32.1 min, 46.0 mg), and 11 (35.7 min, 4.5 mg). Compounds 2 (24.4 min, 3.4 mg) and 7 (28.3 min, 19.0 mg) were acquired from fraction D3-2 by prep-HPLC (MeOH/H₂O = 72:28, v/v). Fr.D4 was first subjected to a Sephadex LH-20 column (CH₃OH/CHCl₃, 1:1, v/v) to yield two subfractions (Fr.D4-1 and Fr.D4-2), the purification of Fr.D4-1 was chromatographed with prep-HPLC (MeOH/H₂O = 67:33, v/v) to yield compounds 4 (21.2 min, 1.5 mg), 5 (24.8 min, 2.4 mg), 12 (26.4 min, 15.0 mg), 13 (32.1 min, 6.8 mg), 14 (27.9 min, 2.1 mg), 15 (36.6 min, 5.5 mg), and 16 (39.1 min, 28.0 mg). Compounds 17 (15.5 min, 3.7 mg) and 18 (20.8 min, 11.0 mg) were obtained from fraction D4-2 by prep-HPLC (MeOH/H₂O = 62:38, v/v). Fr.D5 was partitioned on Sephadex LH-20 and silica gel column chromatography to get four subfractions (Fr. D5-1~Fr. D5-4). Compound 5 (22.2 min, 15.0 mg) was obtained by purifying Fr. D5-2 with semipreparative HPLC (MeOH/H₂O = 68:32, v/v).

3.3.1. Sanghusterol A = (22E,24R,10S)-ergosta-4,6,8(9),22-tetraen-11α-ol (1)

White powder; ${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{20}$ −18.5 (c 0.1, MeOH); UV (MeOH): λmax (log ε) 225 (4.47), 256 (4.77) nm; CD (MeOH): λmax (Δε) 235 (−4.39), 275 (+3.48), 341 (+2.06) nm; IR ν_max 2955, 2926, 1711, 1664, 1465, 1372, 1100, 998, 869 cm⁻¹; ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS: m/z 395.3309 [M + H]⁺ (calcd for C₂₈H₄₃O, 395.3308).

3.3.2. Sanghusterol B = (24R)-ergosta-7,9(11)-diene-3β,5β,6α-triol (2)

White powder; ${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{20}$ +35.8 (c 0.1, MeOH); UV (MeOH): λmax (log ε) 248 (4.84) nm; CD (MeOH): λmax (Δε) 245 (+34.64) nm; IR ν_max 2967, 2880, 1707, 1455, 1376, 1221, 1061, 971 cm⁻¹; ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS: m/z 453.3341 [M + Na]⁺ (calcd for C₂₈H₄₆O₃Na, 453.3339).

3.3.3. Sanghusterol C = (24R)-ergost-7,9(11)-dien-3β,5α,6α-triol (3)

White powder; ${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{20}$ +22.0 (c 0.1, MeOH); UV (MeOH): λmax (log ε) 248 (4.45) nm; CD (MeOH): λmax (Δε) 209 (−5.03), 242 (+14.82) nm; IR ν_max 2953, 2867, 1459, 1376, 1257, 1165, 1033, 983, 840 cm⁻¹; ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS: m/z 453.3341 [M + Na]⁺ (calcd for C₂₈H₄₆O₃Na, 453.3339).

3.3.4. Sanghusterol D = (24R)-6β-methoxyergosta-7-ene-3β,5α-diol (4)

White powder; ${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{20}$ −41.0 (c 0.1, MeOH); UV (MeOH): λmax (log ε) 205 (4.55) nm; CD (MeOH): λmax (Δε) 208 (−67.80) nm; IR ν_max 2951, 2869, 1712, 1664, 1463, 1378, 1157, 1092, cm⁻¹; ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS: m/z 469.3653 [M + Na]⁺ (calcd for C₂₉H₅₀O₃Na, 469.3652).

3.3.5. Sanghusterol E = (22E,24R)-7α-methoxyergosta-8(14),22-diene-3β,5α,6α-triol (5)

White powder; ${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{20}$ −0.40 (c 0.1, MeOH); UV (MeOH): λmax (log ε) 200 (4.89) nm; CD (MeOH): λmax (Δε) 214 (+11.00) nm; IR ν_max 2955, 2872, 1703, 1451, 1378, 1079, 973 cm⁻¹; ¹H and ¹³C NMR data, see Table 1 and Table 2; HRESIMS: m/z 483.3447 [M + Na]⁺ (calcd for C₂₉H₄₈O₄Na, 483.3445).

3.4. ECD Calculation Details

Gaussian 16 software was utilized for the execution of ECD calculations. Conformational optimization was performed in the gas phase via density functional theory (DFT) at the B3LYP/6-31G(d) level. Time-dependent density functional theory (TDDFT) was also adopted for ECD calculations, which were conducted in methanol (MeOH) using the polarizable continuum model (PCM) at the B3LYP/6-311G(d,p) level. ECD spectra were generated via SpecDis 1.7 software [30].

3.5. Anti-Inflammatory Activity

3.5.1. Cell Culture

The RAW 264.7 macrophage cell line was acquired from Procell Life Science & Technology Co., Ltd. (Wuhan, China). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA), which was supplemented with 10% fetal bovine serum (FBS; ABW, Shanghai Nova Pharmaceutical Technology Co., Ltd., Shanghai, China) and 1% penicillin-streptomycin (Beyotime, Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China). All cell lines were maintained at 37 °C in a humidified incubator with 5% CO₂.

3.5.2. Cell Viability

The cytotoxicity of the test compounds on RAW 264.7 macrophages was assessed using a slightly modified version of a previously described protocol [7]. RAW 264.7 cells were seeded into 96-well plates at a density of 8 × 10³ cells per well and incubated for 24 h, followed by exposure to the test compounds at a concentration of 20 µM for another 24 h. Dexamethasone (20 µM) served as the positive control, while 0.1% dimethyl sulfoxide (DMSO) was used as the negative control. After incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well to a final concentration of 0.5 mg/mL. The insoluble formazan crystals were harvested, dissolved in DMSO, and the absorbance was quantified using a microplate reader (BioRad, Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 490 nm.

3.5.3. NO Production in LPS-Induced RAW 264.7 Macrophages

RAW 264.7 macrophages were seeded into 96-well plates at a density of 1.5 × 10⁴ cells per well and incubated for 24 h. The cells were then treated with various concentrations of the test compounds and positive control (0, 0.6125, 1.25, 2.5, 5, 10, and 20 µM) in the presence or absence of lipopolysaccharide (LPS; 1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. The concentration of nitric oxide (NO) in the cell culture supernatant was quantified using a Griess reagent kit (Beyotime, China). Briefly, 100 μL of the culture supernatant from each group and standard curve solutions were added to a 96-well plate, followed by the addition of 100 μL of Griess reagent. The absorbance was detected with a multifunctional microplate reader at 540 nm. All experiments were conducted in triplicate, and the data were expressed as mean ± standard deviation (SD). Dexamethasone was employed as the positive control [7].

3.5.4. Western Blotting

RAW 264.7 cells were seeded into six-well plates at a density of 5 × 10⁵ cells per well and treated with compounds 1, 15, 17, 21, and 25 at different concentrations (0, 5, 10, and 20 μM), followed by stimulation with LPS (1 μg/mL) for 24 h. The cells were collected and lysed on ice for 30 min. The supernatant was obtained by centrifugation at 12,000 rpm and 4 °C for 15 min, and the protein concentration was determined using a BCA protein assay kit (BC3710, Solarbio, Beijing, China). A total of 30 μg of protein from each supernatant sample was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Non-specific binding sites were blocked with TBST buffer containing 5% non-fat milk for 2 h, followed by overnight incubation at 4 °C with primary antibodies. The membranes were then washed three times with TBST buffer and incubated with secondary antibodies at room temperature for 1 h. After three additional washes with TBST buffer, immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) Western blotting kit (Sigma, St. Louis, MO, USA) and quantified using ImageJ 1.52a software. The bands were scanned with a GS-800 scanner and analyzed using a ChemiDoc XRS imaging system (Bio-Rad Life Sciences, Hercules, CA, USA). The primary antibody against β-actin (60008-1-Ig) was purchased from Proteintech (Wuhan, China), whereas COX2 (cat. no. 12282) and iNOS (cat. no. 13120) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA) [7]. All experiments were conducted in triplicate.

3.5.5. Molecular Docking

Molecular docking analyses were performed with PyRx 0.8 software. The X-ray crystal structures of COX-2 (PDB ID: 1CX2) and iNOS (PDB ID: 6KEY) were downloaded from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/, accessed on 31 March 2026). AutoDock 4.2.6 Tools were used to process the PDB structures, which involved adding hydrogen atoms and removing water molecules, and the processed structures were saved in PDBQT format. The 3D structures of the test compounds were optimized and saved in MOL2 format, then subsequently imported into AutoDock Tools. Molecular docking was performed using the AutoDock platform (https://autodock.scripps.edu, accessed on 31 March 2026), and the binding affinities between the compounds and their target proteins were evaluated according to the binding energy values. To visualize the molecular docking results, 2D interaction diagrams of the docking poses were generated using LigPlot+ 2.3.1, and 3D representations of the binding modes were visualized with PyMOL 3.1.

3.6. Statistical Analysis

All data are expressed as mean ± SEM from at least three independent experiments. Statistical significance for the LPS-induced iNOS and COX-2 inhibition assays was evaluated using one-way ANOVA followed by Dunnett’s post hoc test, with the untreated control group as the reference. A p-value < 0.05 was considered statistically significant. Data analysis was performed using GraphPad Prism 9.0.

4. Conclusions

In summary, 26 compounds, including five new steroids (1–5), were isolated from S. vaninii. Their structure was elucidated based on detailed spectroscopic analysis and quantum chemical calculations. Concurrently, compounds 1, 15, 17, 21 and 25 displayed potent inhibitory effects on LPS-induced NO production in RAW264.7 macrophages, with IC₅₀ values of 8.3–14.8 μM. Mechanistically, these compounds dose-dependently downregulated the protein expression levels of iNOS and COX-2, two pivotal mediators of the inflammatory response. These findings validate the traditional medicinal application of S. vaninii and provide a solid phytochemical and pharmacological basis for developing S. vaninii-derived compounds as potential anti-inflammatory agents.